DEVELOPMENT OF NEW MODELS OF OCULAR INFLAMMATION: (1) We continue to collaborate with the group of Dr. Warren Strober (NIAID) on ocular inflammation in NLRP3 knock-in mice. This gene is associated with Muckle-Wells syndrome, which among other pathologies is also associated with inflammation at the ocular surface. Studies with these mice indicate existence of an abnormal response to what we believe to be the normal flora of the ocular surface. These studies may provide new insights into ocular involvement in Muckle-Wells syndrome. (2) A new model of conjunctivitis has been developed by challenging the mucosal tissues of the ocular surface with innate microbial stimuli. Findings reveal that the conjunctiva-associated lymphoid tissue (CALT) contains a variety of leukocytes, among them innate-like lymphocytes (ILCs), gamma-delta T cells and alpha-beta T cells, which respond dynamically. We are defining the response and functionality of these cells in controlling infection and inflammation at the ocular surface. The insights may have important implications on treatment of ocular surface disease. (3) In collaboration with Drs. Daniela Verthelyi and Mohanraj Manangeeswaran of FDA/CBER we developed and studied a model of Zika virus infection of the eye. Areas of retinal degeneration and gliosis persist at 60 days post infection. The model recapitulates the progression of ZIKA infections in patients. It has the potential to help elucidate the mechanisms underlying Zika damage to the eyes and aid in the development of effective therapeutics (Manangeeswaran M et al., submitted for publication). FUNDAMENTAL MECHANISMS IN TOLERANCE, IMMUNITY AND AUTOIMMUNITY TO RETINAL ANTIGENS These studies rely heavily on the disease models developed in our laboratory, experimental autoimmune uveitis (EAU) induced in mice by immunization with the retinal Ag IRBP, and spontaneous uveitis, which develops in R161H mice, which express a transgenic T cell receptor (TCR) specific for the IRBP epitope encoded by residues 161-180, on the B10.RIII background. To better study basic mechanisms, we have backcrossed numerous gene-manipulated strains onto the B10.RIII/R161H background. (1) Recent findings in R161H mice indicated that commensal flora contributes to development of spontaneous uveitis. Mechanistic studies revealed that signaling through the clonotypic TCR in the gut by a non-cognate antigen derived from gut microflora is a necessary (though possibly not sufficient) stimulus, and results in activation of retina-specific T cells in the gut to the Th17 phenotype (Horai et al, Immunity 2015). We are currently analyzing the flora associated with disease and attempting to dissect whether human flora will support development of disease, with the aim of identifying the disease-associated component(s) of the gut flora. (2) The role of various cytokines in pathogenesis and regulation of uveitis is being studied using both the classical and the spontaneous uveitis models, with emphasis on the balance between Th1/Th17 responses and their control by T-regulatory (Treg) cells. Although previous studies indicated a critical role for the Th17 lineage cytokines in pathogenesis of immunization-induced EAU, crossing R161H mice to IFN-g deficient or IL-17 deficient mice unexpectedly revealed a major role for IFN-g in the spontaneous disease. These findings raise the possibility that IL-17 produced by R161H cells in the gut (above), is a marker of the pathogenic cells but not necessarily the pathogenic cytokine itself. The role of other Th17 lineage cytokines, including TNF-, GM-CSF, IL-22 and IL-17F are also being examined. Findings have implicated GM-CSF as a pathogenic cytokine, which may drive disease in the absence of IL-17 (S Bing et al, in preparation). On the other hand, IL-22 appears to have a protective role, part of which may to be due to a neuroprotective effect of IL-22. Since neurons do not seem to express a receptor for IL-22, this is likely an indirect effect that requires other retinal cells (Mattapallil et al, in preparation). Importantly, IL-17 may regulate production of other Th17 lineage-specific cytokines. The cellular and molecular mechanisms are currently being studied. (WP Chong et al., in preparation). Finally, in collaboration with the group of Dr. Charles Egwuagu (NEI, LI, project # EY000350-18) it was found that IL-12p35 is an effective immunoregulatory agent. Treatment with this engineered cytokine induced IL-10 and IL-35-expressing regulatory B cells and T cells, inhibited the expansion and trafficking of pathogenic Th17 and Th1 cells, and ameliorated EAU (Dambuza et al, Nature Communications, in press) and EAE (J Choi et al., Frontiers in Immunology, provisionally accepted). (3) Vitamin (VitA) derivatives are necessary for functional activation of immune cells (published literature). We previously demonstrated the importance of Vitamin A (VitA) and its metabolite, retinoic acid, in ocular immune privilege. Using mice made VitA deficient (VAD), we found that T cell effector function that was acquired before onset of VAD is maintained in the VAD host. These findings may have clinical implications in geographical regions where dietary VitA is limiting. (Horai, Zhou et al, in preparation). EFFECTS OF INNATE IMMUNE RESPONSES ON AUTOIMMUNITY: Cellular and molecular elements of the innate immune system can affect immunopathogenic processes directly as well as indirectly, by affecting adaptive immunity. (1) The transcription factor STAT3 has been defined as necessary for the induction of IL-17 production by adaptive and innate cells. Our recent studies indicate that some types of innate lymphocytes, including NKT cells, innate-like CD8 cells and CD4/CD8 double-negative T cells, are able to produce IL-17 via a pathway that does not require STAT3. In vivo experiments suggest that this pathway may have a function in protecting from infections at the ocular surface. (St. Leger, Hansen et al, in revision). (2) Innate immunity receptors have a major role in controlling susceptibility to autoimmune uveitis. A collaborative study with Dr. Holly Rosenzweig lab at OHSU, Portland, is dissecting the revealed that the SYK/CARD9 signaling pathway is necessary for uveitis and can be driven by fungal cues through the Dectin-2/Card9-coupled pathway in ocular autoimmunity (Brown et al., Clin Exp Immunol.). (3) A collaborative study with Dr. Feng Lin at the Cleveland Clinic, Cleveland, Ohio, revealed that the Complement component C4 regulates T cell responses in a cell-intrinsic fashion (Lin et al., Frontiers in Immunology, in press). THE OCULAR MICROBIOME AND MUCOSAL IMMUNE RESPONSES AT THE OCULAR SURFACE: Mucosal sites such as the intestine, oral cavity, nasopharynx, and vagina all have associated commensal flora. The surface of the eye is also a mucosal site, but proof of a living, resident ocular microbiome remains elusive. We used a mouse model of ocular surface disease to reveal that commensals were present in the ocular mucosa and had functional immunological consequences. We isolated and purified one such candidate commensal, Corynebacterium mastitidis and showed that this organism elicited a commensal-specific interleukin 17 response from T cells in the ocular mucosa that was central to local immunity. The commensal-specfic response drove neutrophil recruitment and the release of antimicrobials into the tears, and protected the eye from pathogenic Candida albicans or Pseudomonas aeruginosa infection. Our findings provide direct evidence that a resident commensal microbiome exists on the ocular surface and identify the cellular mechanisms underlying its effects on ocular immune homeostasis and host defense. (St. Leger et al., Immunity 2017).